Bacteria from the genus Agrobacterium have the ability to transfer specific segments of DNA (T-DNA) to plant cells, where they stably integrate into the nuclear chromosomes. Analyses of plants harbouring the T-DNA have revealed that this genetic element may be integrated at numerous locations, and can occasionally be found within genes. One strategy which may be exploited to identify integration events within genes is to transform plant cells with specially designed T-DNA vectors which contain a reporter gene, devoid of cis-acting transcriptional and translational expression signals (i.e. promoterless), located at the end of the T-DNA. Upon integration, the initiation codon of the promoterless gene (reporter gene) will be juxtaposed to plant sequences. The consequence of T-DNA insertion adjacent to, and downstream of, gene promoter elements may be the activation of reporter gene expression. The resulting hybrid genes, referred to as T-DNA-mediated gene fusions, consist of unknown and thus uncharacterized plant promoters residing at their natural location within the chromosome, and the coding sequence of a marker gene located on the inserted T-DNA (Fobert et al., 1991, Plant Mol. Biol. 17, 837-851).
It has generally been assumed that activation of promoterless or enhancerless marker genes result from T-DNA insertions within or immediately adjacent to genes. The recent isolation of several T-DNA insertional mutants (Koncz et al., 1992, Plant Mol. Biol. 20, 963-976; reviewed in Feldmann, 1991, Plant J. 1, 71-82; Van Lijsebettens et al., 1991, Plant Sci. 80, 27-37; Walden et al., 1991, Plant J. 1: 281-288; Yanofsky et al., 1990, Nature 346, 35-39), shows that this is the case for at least some insertions. However, other possibilities exist. One of these is that integration of the T-DNA activates silent regulatory sequences that are not associated with genes. Lindsey et al. (1993, Transgenic Res. 2, 3347) referred to such sequences as “pseudo-promoters” and suggested that they may be responsible for activating marker genes in some transgenic lines.
Inactive regulatory sequences that are buried in the genome but with the capability of being functional when positioned adjacent to genes have been described in a variety of organisms, where they have been called “cryptic promoters” (Al-Shawi et al., 1991, Mol. Cell. Biol. 11, 4207-4216; Fourel et al., 1992, Mol. Cell. Biol. 12, 5336-5344; Irniger et al., 1992, Nucleic Acids Res. 20, 4733-4739; Takahashi et al., 1991, Jpn J. Cancer Res. 82, 1239-1244). Cryptic promoters can be found in the introns of genes, such as those encoding for yeast actin (Irniger et al., 1992, Nucleic Acids Res. 20, 4733-4739), and a mammalian melanoma-associated antigen (Takahashi et al., 1991, Jpn J. Cancer Res. 82, 1239-1244). It has been suggested that the cryptic promoter of the yeast actin gene may be a relict of a promoter that was at one time active but lost function once the coding region was assimilated into the exon-intron structure of the present-day gene (Irniger et al., 1992, Nucleic Acids Res. 20, 4733-4739). A cryptic promoter has also been found in an untranslated region of the second exon of the woodchuck N-myc proto-oncogene (Fourel et al., 1992, Mol. Cell. Biol. 12, 5336-5344). This cryptic promoter is responsible for activation of a N-myc2, a functional processed gene which arose from retroposition of N-myc transcript (Fourel et al., 1992, Mol. Cell. Biol. 12, 5336-5344). These types of regulatory sequences have not yet been isolated from plants.
Weber et al. (1995, Plant Cell 7:1835-1846) disclose a cDNA sequence of a seed-coat associated invertase. However, all of the cDNA's characterized were found to be expressed in tissues other than the seed-coat, including anthers, cotyledon, stem and root. Furthermore, no promoter was isolated, characterized, or disclosed.
Described herein is the occurrence of seed-coat genes and promoters that have been obtained as a result of differential screening of seed-coat genomic libraries, or generated by tagging with a promoterless GUS (β-glucuronidase) T-DNA vector, or by identification of genes that are highly expressed in the seed-coat or associated tissues. Expression analysis of these DNA's reveal that they are spatially and developmentally regulated in seed coats. Prior to this work, promoters, as well as genes specifically expressed in seed coat tissues had not been isolated or reported. Furthermore, proteins encoded by genes that are expressed within seed-coat, or associated with seed-coat tissues, are also disclosed.